title: Long-term shake suspension and membrane vesicles of medullary thick ascending limb.
abstract: Cultured medullary thick ascending limb (MTAL) cells may lack some of the main carriers of fresh MTAL cells, such as apical Na+-K+(NH4+)-2Cl- cotransporter (BSC-1) and Na+/H+ exchanger (NHE-3). We have developed a technique to maintain rat MTALs several hours in suspension and in a good state of viability. Medullary thick ascending limbs were suspended in a 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium (HDMEM) supplemented with 25 mM HCO3- and gassed with 95% O2/5% CO2; the resulting mixture was placed in a rotary shaking water bath at 37 degrees C for 16 hours. As seen by electron microscopy, MTALs from the HDMEM-suspension retained a virtually normal tubular organization. Na+-K+(NH4+)-2Cl- cotransport activity and NHE consistent with both apical NHE-3 and basolateral NHE-1 activities were underscored both in intact cells by intracellular pH measurements and in a membrane fraction enriched in apical and basolateral membranes by 22Na+ uptake experiments. These results demonstrate that freshly harvested MTALs can be maintained in a well differentiated state for at least 16 hours; this preparation should make long-term in vitro studies of MTAL transport regulations possible.
authors.affiliation: Physiologie et Endocrinologie Cellulaire Rénale, INSERM U. 356, Université Paris 6-Pierre et Marie Curie, and Hôpital Broussais, France.
authors.forename: A
authors.initials: A
authors.lastname: Attmane-Elakeb
authors.forename: V
authors.initials: V
authors.lastname: Sibella
authors.forename: A
authors.initials: A
authors.lastname: Moreau
authors.forename: C
authors.initials: C
authors.lastname: Vernimmen
authors.forename: G
authors.initials: G
authors.lastname: Feldmann
authors.forename: M
authors.initials: M
authors.lastname: Paillard
authors.forename: M
authors.initials: M
authors.lastname: Bichara
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002458:Cell Fractionation; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D008138:Loop of Henle / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D051381:Rats; D017207:Rats, Sprague-Dawley; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase